Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis
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eLife assessment
This important study reveals that the malaria parasite protein PfHO, though lacking typical heme oxygenase activity, is vital for the survival of Plasmodium falciparum. Structural and localization analyses showed that PfHO is essential for apicoplast maintenance, particularly in gene expression and biogenesis, indicating a novel adaptive role for this protein in parasite biology. While the results supporting the claims of the authors are convincing, the lack of data defining a molecular understanding or mechanism of action of the protein in question limits the impact of the study.
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Abstract
Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.
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eLife assessment
This important study reveals that the malaria parasite protein PfHO, though lacking typical heme oxygenase activity, is vital for the survival of Plasmodium falciparum. Structural and localization analyses showed that PfHO is essential for apicoplast maintenance, particularly in gene expression and biogenesis, indicating a novel adaptive role for this protein in parasite biology. While the results supporting the claims of the authors are convincing, the lack of data defining a molecular understanding or mechanism of action of the protein in question limits the impact of the study.
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Reviewer #1 (Public Review):
Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to their disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP reporter fused to the first 83 residues …
Reviewer #1 (Public Review):
Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to their disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP reporter fused to the first 83 residues of PfHO (PfHO N-term-GFP). To investigate the functional importance of PfHO, the authors generated an anhydrotetracycline (aTC) controlled PfHO knockdown strain. Strikingly, the parasites lacking PfHO failed to grow and lost their apicoplast. Finally, by chromatin immunoprecipitation (ChIP), quantitative PCR/RT-PCR, and growth assays, the authors showed that both the cognate N-terminus and HO-like domain were required for PfHO function as an apicoplast DNA interacting protein.
The authors systemically performed multidisciplinary approaches to address this difficult question: what is the function of this enzymatically dead PfHO? I enjoyed reading this manuscript and its thoughtful discussion. This study is not of clinical importance for antimalarial treatments but also deepens our understanding of protein function evolution. While I understand these experiments are challenging to conduct in malaria parasites, the data quality of some of the experiments could be improved. For example, most of the Western blots and Southern blots are not of high quality.
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Reviewer #2 (Public Review):
Summary:
Blackwell et al. investigated the structure, localization, and physiological function of Plasmodium falciparum (Pf) heme oxygenase (HO). Pf and other malaria parasites scavenge and digest large amounts of hemoglobin from red cells for sustenance. To counter the potentially cytotoxic effects of heme, it is biomineralized into hemozoin and stored in the food vacuole. Another mechanism to counteract heme toxicity is through its enzymatic degradation via heme oxygenases. However, it was previously found by the authors that PfHO lacks the ability to catalyze heme degradation, raising the intriguing question of what the physiological function of PfHO is. In the current contribution, the authors determine that PfHO localizes to the apicoplast, determine its targeting sequence, establish the essentiality of …
Reviewer #2 (Public Review):
Summary:
Blackwell et al. investigated the structure, localization, and physiological function of Plasmodium falciparum (Pf) heme oxygenase (HO). Pf and other malaria parasites scavenge and digest large amounts of hemoglobin from red cells for sustenance. To counter the potentially cytotoxic effects of heme, it is biomineralized into hemozoin and stored in the food vacuole. Another mechanism to counteract heme toxicity is through its enzymatic degradation via heme oxygenases. However, it was previously found by the authors that PfHO lacks the ability to catalyze heme degradation, raising the intriguing question of what the physiological function of PfHO is. In the current contribution, the authors determine that PfHO localizes to the apicoplast, determine its targeting sequence, establish the essentiality of PfHO for parasite viability, and determine that PfHO is required for proper maintenance of apicoplasts and apicoplast gene expression. In sum, the authors establish an essential physiological function for PfHO, thereby providing new insights into the role of PfHO in plasmodium metabolism.
Strengths:
The studies are rigorously conducted and the results of the experiments unambiguously support a role for PfHO as being an apicoplast-targeted protein required for parasite viability and maintenance of apicoplasts.
Weaknesses:
While the studies conducted are rigorous and support the primary conclusions, the lack of experiments probing the molecular function of PfHO limits the impact of the work. Nevertheless, the knowledge that PfHO is required for parasite viability and plays a role in the maintenance of apicoplasts is still an important advance.
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Author response:
Public Reviews:
Reviewer #1 (Public Review):
Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to their disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP …
Author response:
Public Reviews:
Reviewer #1 (Public Review):
Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to their disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP reporter fused to the first 83 residues of PfHO (PfHO N-term-GFP). To investigate the functional importance of PfHO, the authors generated an anhydrotetracycline (aTC) controlled PfHO knockdown strain. Strikingly, the parasites lacking PfHO failed to grow and lost their apicoplast. Finally, by chromatin immunoprecipitation (ChIP), quantitative PCR/RT-PCR, and growth assays, the authors showed that both the cognate N-terminus and HO-like domain were required for PfHO function as an apicoplast DNA interacting protein.
The authors systemically performed multidisciplinary approaches to address this difficult question: what is the function of this enzymatically dead PfHO? I enjoyed reading this manuscript and its thoughtful discussion. This study is not of clinical importance for antimalarial treatments but also deepens our understanding of protein function evolution. While I understand these experiments are challenging to conduct in malaria parasites, the data quality of some of the experiments could be improved. For example, most of the Western blots and Southern blots are not of high quality.
We thank the reviewer for the positive comments but are a bit puzzled by the final statement about western and Southern blot quality. We agree that the two anti-PfHO western blots probed with custom antibody (Fig. 3- source data 2 and 8) have substantial background signal in the higher molecular mass region >75 kDa. However, we note that the critical region <50 kDa is clear in both cases and readily enables target band visualization. All other western blots probing GFP or HA epitopes are of high quality with minimal off-target background. We present two Southern blot images. We agree that the signal is somewhat faint for the Southern blot demonstrating on-target integration of the aptamer/TetR-DOZI plasmid (Fig. 3- fig. supplement 4), although we note that the correct band pattern for integration is visible. We also note that the accompanying genomic PCR data is unambiguous. The Southern blot for GFP-DHFRDD incorporation into the PfHO locus (Fig. 3- fig. supplement 1) has clear signal and strongly supports on-target integration. The minor background signal in the lower left region of the image does not extend into nor impact interpretation of correct clonal integration.
Reviewer #2 (Public Review):
Summary:
Blackwell et al. investigated the structure, localization, and physiological function of Plasmodium falciparum (Pf) heme oxygenase (HO). Pf and other malaria parasites scavenge and digest large amounts of hemoglobin from red cells for sustenance. To counter the potentially cytotoxic effects of heme, it is biomineralized into hemozoin and stored in the food vacuole. Another mechanism to counteract heme toxicity is through its enzymatic degradation via heme oxygenases. However, it was previously found by the authors that PfHO lacks the ability to catalyze heme degradation, raising the intriguing question of what the physiological function of PfHO is. In the current contribution, the authors determine that PfHO localizes to the apicoplast, determine its targeting sequence, establish the essentiality of PfHO for parasite viability, and determine that PfHO is required for proper maintenance of apicoplasts and apicoplast gene expression. In sum, the authors establish an essential physiological function for PfHO, thereby providing new insights into the role of PfHO in plasmodium metabolism.
Strengths:
The studies are rigorously conducted and the results of the experiments unambiguously support a role for PfHO as being an apicoplast-targeted protein required for parasite viability and maintenance of apicoplasts.
Weaknesses:
While the studies conducted are rigorous and support the primary conclusions, the lack of experiments probing the molecular function of PfHO limits the impact of the work. Nevertheless, the knowledge that PfHO is required for parasite viability and plays a role in the maintenance of apicoplasts is still an important advance.
We appreciate the positive assessment. We agree that further mechanistic understanding of PfHO function remains a key future challenge. Indeed, we made extensive efforts to unravel PfHO interactions that underpin its critical function. We elucidated key interactions with the apicoplast genome, reliance on the electropositive N-terminus, association with DNA-binding proteins, and a specific defect in apicoplast mRNA levels. The major limitation we faced in further defining PfHO function is the general lack of understanding of apicoplast transcription and broader gene expression. That limitation and the challenges to overcome it go well beyond our study and will require concerted efforts across several manuscripts (likely by multiple groups) to define the mechanistic features of apicoplast gene expression. We look forward to contributing further molecular understanding of PfHO function as broader understanding of apicoplast transcription emerges.
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